Embryonic-fetal erythroid characteristics of a human leukemic cell line.

Benz, Edward J.; Murnane, Mary Jo; Tonkonow, B L; Berman, Brian; Mazur, Eric Michael; Cavallesco, Cesira; Jenko, Trina; Snyder, Edwin L.; Forget, Bernard G.; Hoffman, Ronald

doi:10.1073/pnas.77.6.3509

Cited by 149 publications

(74 citation statements)

References 19 publications

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“…1b, lanes 2 and 4). In K562 cells, the ε-globin gene is expressed (3,7,10); accordingly, the sense transcripts of the gene produced the expected amplification band of 84 bp (Fig. 1b, lane 5).…”

Section: Resultsmentioning

confidence: 99%

Transcription of the HS2 Enhancer toward a cis-Linked Gene Is Independent of the Orientation, Position, and Distance of the Enhancer Relative to the Gene

Kong

Bohl

et al. 1997

Molecular and Cellular Biology

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The locus control region (LCR) of the human ␤-globin gene domain is defined by four erythroid-cell-specific DNase I-hypersensitive (HS) sites, HS1, -2, -3, and -4, located 50 to 70 kb upstream of the ␤-globin gene in a transcriptional direction: 5Ј HS4-HS3-HS2-HS1-//-ε-G ␥-A ␥-␦-␤ 3Ј (17,20,37,50). The LCR has been shown by studies of natural deletions of ␥␦␤-thalassemia (8,11,24) and by gene transfer experiments (14,18,20,51) to be indispensable for erythroid-cell-specific and high-level transcription of cis-linked globin genes and transgenes. The mechanism by which the LCR regulates transcription of the distant embryonic ε-, fetal ␥-, and adult ␤-globin genes at the respective developmental stages is not fully understood.In previous studies examining the mechanism of LCR function, a number of laboratories have investigated the ability of the individual HS sites to activate the transcription of cislinked genes in cell lines and transgenic mice. Among the LCR HS sites, HS2 has been found to possess developmental-stageindependent enhancer function (52). It is capable of stimulating the transcription of embryonic ε-, fetal ␥-, and adult ␤-globin genes in erythroid cells at the corresponding developmental stages (9,28,34,42,45,47) and may therefore constitute a major functional component of the LCR. However, a recent targeted deletion study shows that deletion of the HS2 enhancer from the murine LCR generated only mild effects on the expression of the ␤-like globin genes (15), suggesting that HS2 is not essential for LCR function and that LCR function may be due to the contribution of the other HS sites. Surprisingly, similar targeted deletion of HS3 also did not cause significant changes in the expression of the ␤-like globin genes (22). These findings suggest that LCR function is due not to the contribution of any specific HS site but to a series of interactions among its functional components, including the enhancer elements underlying the HS sites.In the above-described targeted deletion studies, deleting the HS2 or the HS3 sequence from the endogenous murine LCR and inserting in its place an independent transcription unit-a neomycin-resistant gene driven by a strong promoter-has been found to disrupt LCR function and cause severe anemia in and the deaths of homozygous transgenic mice (15,21). In the human ␤-LCR, inserting an independent transcription unit at a location between the HS2 enhancer and the downstream globin genes also disrupted LCR function (23) and caused the distantly downstream ␤-globin gene to be transcriptionally turned off. The mechanism by which the transcription of a foreign gene within the LCR might disrupt LCR function is not clearly understood (21).In an attempt to delineate the functional mechanism of the HS2 enhancer and ultimately of the LCR, we have previously analyzed the transcriptional status of the HS2 enhancer in transfected recombinant chloramphenicol acetyltransferase (CAT) plasmids (53). This earlier study showed that the transfected HS2 enhancer is itself transcribed in eryt...

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Section: Resultsmentioning

confidence: 99%

Transcription of the HS2 Enhancer toward a cis-Linked Gene Is Independent of the Orientation, Position, and Distance of the Enhancer Relative to the Gene

Kong

Bohl

et al. 1997

Molecular and Cellular Biology

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“…The ␤-and ␦-␤-EGFP EBAC constructs produced not only the lowest levels of transfection but also the lowest level of EGFP expression, whereas the G ␥-, A ␥-, and ⑀-EGFP ␤-globin EBACs resulted in the highest levels of transfection and EGFP expression, which is in line with the embryonic phenotype of globin gene expression of K562 cells. 44,45 We have previously demonstrated that transient transfection of MEL cells with pEBAC/148␤-EGFP enabled the identification of clones expressing EGFP under the regulatory elements of the ␤-globin gene. 44 However, the efficiency of transfection was very low, making it difficult to perform functional studies.…”

Section: Transient Transfection Studies With Egfp-modified Globin Ebamentioning

confidence: 99%

“…44,45 We have previously demonstrated that transient transfection of MEL cells with pEBAC/148␤-EGFP enabled the identification of clones expressing EGFP under the regulatory elements of the ␤-globin gene. 44 However, the efficiency of transfection was very low, making it difficult to perform functional studies. In this study, we have generated stable clones of MEL cells expressing EBNA-1 (MEL-EBNA1) and demonstrated a similar increase in transfection efficiency with our EBAC constructs, as with K562-EBNA1 cells (data not shown).…”

Section: Transient Transfection Studies With Egfp-modified Globin Ebamentioning

confidence: 99%

Development of sensitive fluorescent assays for embryonic and fetal hemoglobin inducers using the human β-globin locus in erythropoietic cells

Vadolas

Wardan

Orford

et al. 2002

Blood

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Reactivation of fetal hemoglobin genes has been proposed as a potential therapeutic procedure in patients with ␤-thalassemia, sickle cell disease, or other ␤-hemoglobinopathies. In vitro model systems based on small plasmid globin gene constructs have previously been used in human and mouse erythroleukemic cell lines to study the molecular mechanisms regulating the expression of the fetal human globin genes and their reactivation by a variety of pharmacologic agents. These studies have led to great insights in globin gene regulation and the identification of a number of potential inducers of fetal hemoglobin. In this study we describe the development of enhanced green fluorescence protein (EGFP) reporter systems based on bacterial artificial chromosomes (EBACs) to monitor the activity of the ⑀-, G ␥-, A ␥-, ␦-, and ␤-globin genes in the ␤-globin locus. Additionally, we demonstrate that transfection of erythroleukemia cells with our EBACs is greatly enhanced by expression of EBNA1, which also facilitates episomal maintenance of our constructs in human cells. Our studies in human cells have shown physiologically relevant differences in the expression of each of the globin genes and also demonstrate that hemin is a potent inducer of EGFP expression from EGFPmodified ⑀-, G ␥-, and A ␥-globin constructs. In contrast, the EGFP-modified ␦-and ␤-globin constructs consistently produced much lower levels of EGFP expression on hemin induction, mirroring the in vivo ontogeny. The EGFP-modified ␤-globin eukaryotic BAC (EBAC) vector system can thus be used in erythroleukemia cells to evaluate induction of the ⑀-and ␥-globin genes from the intact human ␤-globin locus. Introduction␤-Thalassemia and sickle cell disease are among the most common inherited genetic disorders in the world. In ␤-thalassemia, unpaired ␣-globin chains accumulate and precipitate within erythroid cells, resulting in red cell damage, hemolysis, ineffective erythropoiesis, and anemia. In sickle cell disease, intracellular accumulation of polymerized hemoglobin causes sickling of red blood cells and vascular occlusion. Amelioration of clinical symptoms associated with these hemoglobinopathies has been reported in patients with elevated levels of ␥-globin chain synthesis. In ␤-thalassemia, the presence of ␥-globin inhibits the precipitation of unpaired ␣-globin through the formation of fetal hemoglobin (HbF, ␣ 2 ␥ 2, ), whereas in sickle cell disease the presence of ␥-globin inhibits the polymerization of sickle hemoglobin (␣ 2 ␤ 2 s ) via the formation of mixed hemoglobin tetramers (␣ 2 ␤ s ␥) and the maintenance of a higher oxygen tension in the capillaries. 1 A diverse group of genetic mutations termed hereditary persistence of fetal hemoglobin (HPFH) are associated with high levels of HbF in adult life. Coexistence of HPFH with homozygous ␤-thalassemia or sickle cell disease often results in complete phenotypic complementation of the disease. 2 Many of the HPFH mutations are the result of deletions or point mutations in the ␤-globin locus. 3 However, a ...

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“…As EGFP expression is driven from the b-globin promoter in clones 3-6, rather than the g-globin promoter as in clones 1 and 2, this is in line with the embryonic phenotype of globin gene expression observed in K562 cells. 36,37 Treatment with either hemin (0-50 mM) or cisplatin (0-15 mM) also caused a significant dose-dependent shift in the level of EGFP expression in clones 1-2, with a similar increase in the level of dsRed expression observed for clones 3-6 (Figures 7d and e). Overall, hemin was more effective in inducing reporter gene expression in all six clones, with clone 6 showing the highest level of reporter gene induction for both compounds.…”

Section: Reporter Gene Expressionmentioning

confidence: 62%

Site-specific, Rep-mediated integration of the intact β-globin locus in the human erythroleukaemic cell line K562

et al. 2008

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The stable, regulated and tissue-specific expression of a therapeutic transgene can be best achieved by the transfer of a complete genomic locus, which will include the shortand long-range regulatory elements that are critical for the accurate control of gene expression. However, when techniques that rely on the random integration of exogenous DNA into the human genome are used for gene transfer, the risk of insertional mutagenesis remains a major issue. Using components derived from the adeno-associated virus (AAV), we have successfully targeted the integration of 200 kb bacterial artificial chromosomes containing the entire b-globin locus into the AAVS1 site on human chromosome 19. We show that transient expression of the AAV Rep proteins in K562 cells facilitated site-specific transgene integration in 17% (6 of 36) of all analysed integration sites. Southern blot analysis revealed the locus had integrated into AAVS1 as an intact, functional unit in five of the six clones generated. Furthermore, each of the site-specific integrants exhibited sustained and appropriately regulated transgene gene expression over a period of 8 months of continuous culture in the absence of selective pressure. We anticipate that the approach developed in this study may be suitable for facilitating targeted integration of intact genomic loci in adult and embryonic stem cells, and therefore provide a powerful tool not just for functional studies but in establishing model systems for the ex vivo correction of genetic disorders.

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Embryonic-fetal erythroid characteristics of a human leukemic cell line.

Cited by 149 publications

References 19 publications

Transcription of the HS2 Enhancer toward a cis-Linked Gene Is Independent of the Orientation, Position, and Distance of the Enhancer Relative to the Gene

Transcription of the HS2 Enhancer toward a cis-Linked Gene Is Independent of the Orientation, Position, and Distance of the Enhancer Relative to the Gene

Development of sensitive fluorescent assays for embryonic and fetal hemoglobin inducers using the human β-globin locus in erythropoietic cells

Site-specific, Rep-mediated integration of the intact β-globin locus in the human erythroleukaemic cell line K562

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